Abstract BACKGROUND The first case of glyphosate resistance was reported in a capeweed population from Western Australia in our previous study. This current study investigates the resistance mechanisms in the population. RESULTS Target‐site EPSPS gene sequencing revealed two partial sequences of the EPSPS transcripts (1001 bp and 998 bp), and the full‐length sequence (1551 bp) containing the 1001‐bp transcript was cloned as it was found in the resistant plants. A known resistance‐endowing target‐site mutation in the 1551‐bp transcript was identified in the resistant plants, resulting in the Pro‐106‐Ser substitution. The subpopulation derived from these mutant plants exhibited >10‐fold resistance to glyphosate compared to the susceptible population. Additionally, the EPSPS gene (1551 bp) was constitutively expressed at a higher level (4.3‐fold) in the resistant than in the susceptible populations. However, 14 C‐glyphosate foliar uptake was similar with no visual difference in 14 C‐glyphosate translocation from leaves to other parts of a plant, between the resistant and susceptible population. CONCLUSION Glyphosate resistance in the studied population is associated with both a target‐site mutation (Pro‐106‐Ser) and increased EPSPS gene expression. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.